Polycystin-2 (PC2), encoded by the PKD2 gene, is mutated in 10-15% of autosomal dominant polycystic kidney disease (ADPKD) patients. PC2 is a Ca(2+)-permeable nonselective cation channel and is present in kidney and many other organs. Likewise, PKD2-mutated patients and mice exhibit extrarenal abnormalities. In comparison with cysts in the kidney, liver, and pancreas, abnormalities in the heart, brain, and vascular vessels are less understood. In particular, roles of PC2 in muscle and endothelia remain largely unknown. In the present study, using a yeast two-hybrid screening, we discovered that the PC2 carboxyl terminal domain (D682-V968) interacts with the cardiac troponin I, an important regulatory component of the actin microfilament in cardiac muscle cells. This interaction was demonstrated by GST pull-down and microtiter binding assays. Dose-dependent binding between PC2 and troponin I followed a Michaelis-Menten relationship, indicating a 1:1 binding stoichiometry. The interacting domains were located to the R872-H927 segment of PC2 and the M1-V107 and K106-L158 segments of troponin I. Co-immunoprecipitation experiments demonstrated that the cardiac and two skeletal isoforms of troponin I were all associated with PC2, when coexpressed in mouse fibroblast NIH 3T3 cells and Xenopus oocytes. Furthermore, reciprocal co-immunoprecipitation verified the interaction between the native polycystin-2 and troponin I in human adult heart tissues. This study thus provides new evidence for a direct attachment of PC2 to the actin microfilament network, in addition to the recently identified association between PC2 and trypomyosin-1. Troponin I functions as an inhibitory subunit of the troponin complex for calcium-dependent regulation of muscle contraction and as an inhibitor of angiogenesis seen in ADPKD. It is possible that altered interaction due to pathogenic polycystin-1 or -2 mutations can account for angiogenesis in ADPKD and may be corrected to some extent by exogenous troponin I.